Control of plant pests using rna molecules

Abstract

Disclosed are double stranded RNA (dsRNA) molecules that are toxic to insect pests. In particular, interfering RNA molecules capable of modulating expression of a pest insect target gene and that are toxic to the insect pest are provided. Further, methods of making and using the interfering RNA, for example as the active ingredient in an insecticidal composition or in a transgenic plant, to confer protection from insect damage are disclosed.

What is claimed is: 
     
         1 . A double stranded RNA (dsRNA) molecule comprising a sense strand and an antisense strand, wherein a nucleotide sequence of the antisense strand is complementary to a portion of a mRNA polynucleotide transcribable from a coleopteran insect Rpt5 gene that comprises a Rpt5 coding sequence i) having from at least about 90% identity to at least about 99% identity to any of SEQ ID NOs: 8; or ii) comprises any of SEQ ID NOs: 8, and wherein the dsRNA molecule is toxic to at least a coleopteran insect pest. 
     
     
         2 . The dsRNA of  claim 1 , wherein the portion of the mRNA polynucleotide comprises i) at least 19 consecutive nucleotides of any of SEQ ID NOs:29-37; ii) from at least 20 or 21 consecutive nucleotides to at least 600 consecutive nucleotides of any of SEQ ID NOs:29-37; or iii) comprises any of SEQ ID NOs:20-28. 
     
     
         3 . The dsRNA of  claim 2 , wherein the antisense strand comprises the complement of i) at least 19 consecutive nucleotides of any of SEQ ID NOs:29-37; ii) from at least 20 or 21 consecutive nucleotides to at least 600 consecutive nucleotides of any of SEQ ID NOs:29-37; or iii) any of SEQ ID NOs:20-28. 
     
     
         4 . The dsRNA molecule of  claim 1 , wherein the nucleotide sequence of the sense strand is substantially or fully complementary to the nucleotide sequence of the antisense strand. 
     
     
         5 . The dsRNA molecule of  claim 1 , wherein the dsRNA is a short hairpin RNA (shRNA) molecule. 
     
     
         6 . The dsRNA molecule of  claim 1 , wherein the coleopteran insect pest is selected from the group consisting of  Melighethes aeneus, Sitophilus oryzae, Sitophilus granaries, Ceutorhynchus assimilis, Rhyzopertha dominica, Phyllotreta nemorum, Phyllotreta striolata, Psylliodes chrysocephala  and  Diabrotica virgifera.    
     
     
         7 . A nucleic acid molecule encoding at least one strand of the dsRNA molecule of  claim 1 . 
     
     
         8 . A recombinant vector comprising a regulatory sequence operably linked to the nucleic acid molecule of  claim 7 . 
     
     
         9 . A bacteria that comprises the recombinant vector of  claim 8 . 
     
     
         10 . An insecticidal composition comprising the dsRNA of  claim 1  and an acceptable agricultural carrier. 
     
     
         11 . The insecticidal composition of  claim 10 , comprising at least a second insecticidal agent. 
     
     
         12 . The insecticidal composition of  claim 11 , wherein the second insecticidal agent is a biological agent or a chemical agent. 
     
     
         13 . The insecticidal composition of  claim 12 , wherein i) the biological agent is a  Bacillus thuringiensis  insecticidal protein, a  Bacillus cereus  insecticidal protein, a  Xenorhabdus  spp. spp. insecticidal protein, a patatin, a protease, a protease inhibitor, a urease, an alpha-amylase inhibitor, a pore-forming protein, a lectin, an engineered antibody or antibody fragment, or a chitinase; or ii) the chemical agent is a carbamate, a pyrethroid, an organophosphate, a friprole, a neonicotinoid, an organochloride, a nereistoxin, or a combination thereof. 
     
     
         14 . A method of controlling at least a coleopteran insect pest comprising contacting said insect pest with a dsRNA comprising a sense strand and an antisense strand, wherein a nucleotide sequence of the antisense strand is complementary to a portion of a mRNA polynucleotide transcribable from a coleopteran insect Rpt5 gene that comprises a Rpt5 coding sequence i) having from at least about 90% identity to at least about 99% identity to any of SEQ ID NOs: 8; or ii) comprises any of SEQ ID NOs:8, and wherein the dsRNA molecule is toxic to at least a coleopteran insect pest. 
     
     
         15 . The method of  claim 14  wherein i) the portion of the mRNA polynucleotide comprises a) at least 19 consecutive nucleotides of any of SEQ ID NOs:29-37; b) from at least 20 or 21 consecutive nucleotides to at least 600 consecutive nucleotides of any of SEQ ID NOs:29-37; or c) comprises any of SEQ ID NOs:20-28; or ii) the antisense strand comprises the complement of a) at least 19 consecutive nucleotides of any of SEQ ID NOs:29-37; b) from at least 20 or 21 consecutive nucleotides to at least 600 consecutive nucleotides of any of SEQ ID NOs:29-37; or c) any of SEQ ID NOs:20-28. 
     
     
         16 . The method of  claim 14 , wherein contacting comprises: a) applying a composition comprising the dsRNA molecule or a nucleic acid molecule encoding the dsRNA molecule or a bacteria comprising the dsRNA molecule or the nucleic acid molecule encoding the dsRNA molecule to a seed or plant, or part thereof, wherein the insect pest feeds on the seed, the plant, or a part thereof; or b) planting a transgenic seed capable of producing a transgenic plant that expresses the dsRNA molecule or a nucleic acid molecule encoding the dsRNA, wherein the pest insect feeds on the transgenic plant, or part thereof. 
     
     
         17 . The method of  claim 16 , wherein the coleopteran insect pest is selected from the group consisting  Melighethes aeneus, Sitophilus oryzae, Sitophilus granaries, Ceutorhynchus assimilis, Rhyzopertha dominica, Phyllotreta nemorum, Phyllotreta striolata, Psylliodes chrysocephala  and  Diabrotica virgifera.    
     
     
         18 . A method of controlling at least a coleopteran insect pest comprising contacting the coleopteran insect pest with a nucleic acid molecule that is or is capable of producing the dsRNA molecule of  claim 1  for inhibiting expression of a Rpt5 target gene in the coleopteran insect pest, and contacting the coleopteran insect pest with at least a second insecticidal agent for controlling a coleopteran insect pest. 
     
     
         19 . The method of  claim 18 , wherein the second insecticidal agent is a biological agent or a chemical agent. 
     
     
         20 . The method of  claim 19 , wherein i) the biological agent is a  Bacillus thuringiensis  insecticidal protein, a  Bacillus cereus  insecticidal protein, a  Xenorhabdus  spp. insecticidal protein, a  Photorhabdus  spp. insecticidal protein, a  Brevibacillus laterosporous  insecticidal protein, a  Lysinibacillus sphearicus  insecticidal protein, a  Chromobacterium  spp. insecticidal protein, a  Yersinia entomophaga  insecticidal protein, a  Paenibacillus popiliae  insecticidal protein, or a  Clostridium  spp. insecticidal protein, a patatin, a protease, a protease inhibitor, a urease, an alpha-amylase inhibitor, a pore-forming protein, a lectin, an engineered antibody or antibody fragment, or a chitinase; or ii) the chemical agent is a carbamate, a pyrethroid, an organophosphate, a friprole, a neonicotinoid, an organochloride, a nereistoxin, or a combination thereof. 
     
     
         21 . A method of identifying a Rpt5 gene in an insect pest for interfering RNA targeting, said method comprising the steps of: a) isolating nucleic acid from an insect pest; b) amplifying an orthologous Rpt5 target gene from the nucleic acid with a pair of primers comprising nucleotide sequences selected from SEQ ID NOs:38-55; c) identifying a sequence of an orthologous Rpt5 target gene; d) producing a dsRNA molecule, wherein the dsRNA molecule comprises a sense strand and an antisense strand, wherein a nucleotide sequence of the antisense strand is complementary to a portion of a mRNA polynucleotide transcribable from the insect pest Rpt5 gene that comprises a Rpt5 coding sequence, and e) testing the dsRNA molecule of step d) for insecticidal activity against the insect pest.